Analysis of age-associated alternation of SCSA sperm DNA fragmentation index and semen characteristics of 1790 subfertile males in China.

BACKGROUND: It has been identified that incidence of infertility was about 20% among couples worldwide, about 50% caused by male elements. However, conventional semen laboratory detections could not handle clinical needs, which led to more comprehensive parameters for male fertility evaluation. We aimed to investigate the clinical relationship of age-linked changes and the sperm chromatin structure assay (SCSA) sperm DNA fragmentation index (DFI), and routine semen characteristics among subfertile Chinese males. METHODS: 1790 clinical semen specimens were enrolled from February 2018 to October 2019. Clinical and laboratory data including routine semen analyses, sperm DFI, and sperm morphology were collected and showed age-related alterations in semen parameters. RESULTS: Our results, displayed an increase in sperm DFI with age, were demonstrated in three age-groups, particularly within the ≥35-year cohort. There were positive and inverse correlations of sperm DFI with abnormal semen characteristics and with normal morphological parameters, respectively. Furthermore, age, sperm morphology, concentration, and progressive motility, immotile sperm percentage, semen volume, sperm survival, and high acridine orange DNA stainability (indicating immature forms) were found to be independent risk factors affecting sperm DNA integrity. Likewise, men aged ≥35 years had a higher sperm DFI than did normozoospermic men in the overall cohort. Routine semen characteristics, sperm DFI, and morphology tended to alter with age. CONCLUSIONS: The SCSA sperm DFI showed the greatest clinical application in the assessment of male fertility in this study, which should help infertility clinics decide on reproductive options for the treatment of older infertile couples.

Tumor suppressive microRNA-200a inhibits renal cell carcinoma development by directly targeting TGFB2.

A large body of evidence indicates that microRNAs play a critical role in tumor initiation and progression by negatively regulating oncogenes or tumor suppressor genes. Here, we report that the expression of miR-200a was notably downregulated in 45 renal cell carcinoma (RCC) samples. Restoration of miR-200a suppressed cell proliferation, migration, and invasion in two RCC cell lines. Furthermore, we used an epithelial-to-mesenchymal transition PCR array to explore the putative target genes of miR-200a. By performing quantitative real-time PCR, ELISA, and luciferase reporter assays, transforming growth factor beta2 (TGFB2) was validated as a direct target gene of miR-200a. Moreover, siRNA-mediated knockdown of TGFB2 partially phenocopied the effect of miR-200a overexpression. These results suggest that miR-200a suppresses RCC development via directly targeting TGFB2, indicating that miR-200a may present a novel target for diagnostic and therapeutic strategies in RCC.

Expressions of miR-15a and its target gene HSPA1B in the spermatozoa of patients with varicocele.

Hyperthermia and oxidative stresses are the two central elements contributing to varicocele-related sperm damage. Growing evidence indicates that microRNAs (miRNAs) are involved in the regulation of the heat and oxidative stress responses. In this study, we analyzed the expressions of several stress-related miRNAs in the sperm and found that the expression of miR-15a was significantly decreased in patients with varicocele compared with the control. Furthermore, miR-15a repressed the expression of HSPA1B, which is a typical stress-induced chaperone protein, through directly binding its 3'-UTR. The expressions of miR-15a and HSPA1B exhibited an inverse correlation in sperm. Our results provide a valuable insight into the varicocele-related sperm impairment and male infertility, and may help to develop potential therapeutic targets and novel biomarkers for male infertility.

Morphological and Molecular Analysis Identified a Subspecies of Crassostrea ariakensis (Fujita, 1913) along the Coast of Asia.

Crassostrea ariakensis (Fujita, 1913) is one of the most important economic and ecological oysters that is naturally distributed along the coast of Asia, separated by the Yangtze River estuary. They are usually compared as different populations, while there is no consensus on whether C. ariakensis in northern and southern areas should be considered as two species or subspecies. Here, we analyzed morphological characteristics, COI, 16s rRNA, mitogenome sequences, and species delimitation analysis (ASAP and PTP) to resolve the intraspecific taxonomic status of the C. ariakensis. Phylogenetic and ASAP analysis highlight that C. ariakensis was divided into N-type and S-type. PTP was unable to differentiate between the two types of C. ariakensis. The divergence time of N-type and S-type C. ariakinsis is estimated to be 1.6 Mya, using the relaxed uncorrelated lognormal clock method. Additionally, significant morphological differences exist between the two groups in terms of the adductor muscle scar color. Despite these differences, the COI (0.6%) and 16S rRNA (0.6%) genetic distance differences between N-type and S-type C. ariakensis has not yet reached the interspecific level. These results suggest that N-type and S-type C. ariakensis should be treated as different subspecies and renamed as C. ariakensis ariakensis subsp. nov and C. ariakensis meridioyangtzensis subsp. nov.

Expression profiling of circular RNA reveals a potential miR-145-5p sponge function of circ-AFF2 and circ-ASAP1 in renal cell carcinoma.

OBJECTIVES: Circular RNAs (circRNAs) are involved in carcinogenesis, though their expression profile in renal cell carcinoma (RCC) is uncharacterized. The tumor suppressor gene miR-145-5p is expressed in RCC tissues, but its relationship with circRNAs is unknown. Thus, we aimed to identify differentially expressed circRNAs in RCC tissues and to explore the interaction between these circRNAs and miR-145 in the development of RCC. METHODS: We performed high-throughput sequencing and bioinformatics analyses to examine the expression pattern of circRNAs in RCC. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were used to functionally annotate differentially expressed circRNAs. Quantitative real-time polymerase chain reaction (qRT-PCR) was used for sequence verification. Small interfering RNAs were employed to investigate the function and mechanism of circRNAs in RCC. The relationship between miR-145-5p and circRNAs was confirmed using luciferase, RNA immunoprecipitation (RIP), and biotin-coupled probe RNA pull-down assays. RESULTS: Fifty-three circRNAs were significantly and differentially expressed in RCC compared to normal control tissue. Bioinformatic analyses indicated that two significantly upregulated circRNAs, circ-AFF2 and circ-ASAP1, had sequences corresponding to miR-145 response elements. Consistently, the luciferase reporter, RIP, and biotin-coupled probe RNA pull-down assays showed that circ-AFF2 and circ-ASAP1 may repress miR-145 by acting as sponges. circ-AFF2 and circ-ASAP1 were highly expressed in RCC patient-derived tumor samples; their overexpression correlated with poor prognosis and low miR-145 levels. Knockdown of circ-AFF2 or circ-ASAP1 in RCC cell lines inhibited proliferation, underscoring their oncogenic function. A circRNA-miRNA network was constructed for RCC using the differentially expressed circRNAs and projected miRNAs. Candidate genes were verified by RT-qPCR and western blot, indicating that circ-AFF2 and circ-ASAP1 may be connected to RCC proliferation and metastasis. CONCLUSION: circ-AFF2 and circ-ASAP1 were upregulated in RCC and likely promote tumor progression by sponging miR-145. Therefore, both circRNAs should be investigated further as potential diagnostic and therapeutic targets for RCC.

Identification of miR-508-3p and miR-509-3p that are associated with cell invasion and migration and involved in the apoptosis of renal cell carcinoma.

MicroRNAs (miRNAs) have emerged as powerful regulators of multiple processes linked to human cancer, including cell apoptosis, proliferation and migration, suggesting that the regulation of miRNA function could play a critical role in cancer progression. Recent studies have found that human serum/plasma contains stably expressed miRNAs. If they prove indicative of disease states, miRNAs measured from peripheral blood samples may be a source for routine clinical detection of cancer. Our studies showed that both miR-508-3p and miR-509-3p were down-regulated in renal cancer tissues. The level of miR-508-3p but not miR-509-3p in renal cell carcinoma (RCC) patient plasma demonstrated significant differences from that in control plasma. In addition, the overexpression of miR-508-3p and miR-509-3p suppressed the proliferation of RCC cells (786-0), induced cell apoptosis and inhibited cell migration in vitro. Our data demonstrated that miR-508-3p and miR-509-3p played an important role as tumor suppressor genes during tumor formation and that they may serve as novel diagnostic markers for RCC.

[Effects of cyanate on pulmonary epithelial cells and pulmonary function in mice].

OBJECTIVE: To investigate the injury of cyanate on the pulmonary function and morphology of C57/BL6N mice. METHODS: Forty male C57/BL6N mice were randomly divided into two groups: normal control group (20 mice) and cyanate group (20 mice). Mice were exposed to 100 mmol/L cyanate feeding for 4 weeks, and pulmonary Raw (Resistance in Air Way) was measured at the beginning and end of the experiment. The mice were sacrificed at the end of the fourth week of the experiment, and the lung tissues were collected for pathological observation and molecular detection of E-Cadherin and Fibronectin. Well-growing A549 cells in logarithmic growth phase were treated with cyanate at the concentrations of 0, 0.25, 0.5 and 1 mmol/L for 24 h, and the cell viability was detected by CCK8 method; reactive oxygen species ROS fluorescent probe (DCFH-DA) was used to detect the changes of ROS levels, and expressions of E-Cadherin and Fibronectin in cells and pulmonary tissues were detected by Western blot. RESULTS: At the beginning of the experiment, the pulmonary airway resistance values of the mice in the normal control group and the cyanate group were (1.82±0.76)cmH2O/(L·s) and (1.85±0.78)cmH2O/(L·s), respectively, with no significant difference. Four weeks later, the pulmonary airway resistance value of mice in the cyanate group was increased to (4.86±0.87)cmH2O/(L·s) (P＜0.01). The HE staining showed that, compared with the normal control group, the injured alveolar structure, the thickened tracheal wall and the significantly proliferated pulmonary interstitial tissue were observed in the cyanate group. The Masson staining showed that elastic fibers were deposited around the trachea of mice in the cyanate group. The results of CCK8 assay for the viability of A549 cells showed that 0.5 mmol/L cyanate exposure could reduce the viability (P＜0.01). The immunofluorescence staining showed that cyanate could increase ROS level in A549 cells by producing green fluorescence in a concentration-dependent manner. The results of Western blotting showed that 0.5 mmol/L of cyanate treatment on A549 cells could reduce the expression of E-Cadherin (P＜0.01) with increasing concentration of cyanate. The expression level of Fibronectin in A549 cells was increased with the increasing cyanate concentration, and there was a significant difference (P＜0.01) on 1 mmol/L cyanate. Western blot results of lung showed the decreasing expression of E-Cadherin (P＜0.01) and increasing expression of Fibronectin (P＜0.01) in cyanate mice. CONCLUSION: Pathological concentrations of cyanate can induce the proliferation of pulmonary interstitial tissue, fibrous deposition, and increased pulmonary airway resistance in mice, which may be related to damaged pulmonary epithelial cell viability, enhanced ROS production, and induced pathologic changes of extracellular matrix by cyanate.

MicroRNA­205 promotes the tumorigenesis of nasopharyngeal carcinoma through targeting tumor protein p53-inducible nuclear protein 1.

Nasopharyngeal carcinoma (NPC) is a common type of cancer in southern China, miRNAs have been shown to be involved in the tumorigenesis of multiple cancer types. The present study aimed to explore the potential role of miR­205 in NPC. Reverse transcription quantitative polymerase chain reaction was used to determine the expression levels of miR­205 in 20 fresh NPC specimens and 20 normal nasopharyngeal tissues. The function of miR­205 in the proliferation, migration, invasion and apoptosis of NPC­derived cells was detected by MTT assay, colony formation assay, wound healing assay, Transwell assay and flow cytometry. Furthermore, a target gene of miR­205 was identified using the luciferase reporter assay. The expression of miR­205 was increased in NPC tissues compared with that in normal tissues. Overexpression of miR­205 was found to promote the proliferation, migration and invasion of NPC­derived cells, while apoptosis was suppressed. Tumor protein p53-inducible nuclear protein 1 was identified as a target gene of miR­205. Overall, the present study demonstrated that miR­205 may function as an oncogene in NPC tumorigenesis.

miR-145 functions as tumor suppressor and targets two oncogenes, ANGPT2 and NEDD9, in renal cell carcinoma.

PURPOSE: Abnormal expression of miRNAs is closely related to a variety of human cancers. The purpose of this study is to identify new tumor suppressor miRNA and elucidate its physiological function and mechanism in renal cell carcinoma (RCC). METHODS: The expression of miR-145 in 45 RCC and adjacent normal tissues was performed by quantitative RT-PCR. Cell proliferation, migration, invasion, apoptosis and cycle assays were carried out for functional analysis after miR-145 transfection. Two target genes of miR-145 were identified by luciferase reporter assay. The altered expression of 84 epithelial to mesenchymal transition (EMT)-related genes after miR-145 transfection was detected by RT(2) Profiler EMT PCR array. RESULTS: The expression of miR-145 was downregulated in RCC compared to their normal adjacent tissues. Restoring miR-145 expression in RCC cell lines dramatically suppressed cell proliferation, migration and invasion, and induced cell apoptosis and G2-phase arrest. We further validated those miR-145 targets two oncogenes, ANGPT2 and NEDD9 in RCC. In addition, miR-145 was found to regulate numerous genes involved in the EMT. CONCLUSIONS: These findings demonstrate that miR-145 functions as tumor suppressor in RCC, suggesting that miR-145 may be a potential therapeutic target for RCC.

[Idenfication of microRNAs profiles in nasopharyngeal carcinoma].

OBJECTIVE: To filtrate and prove the different microRNAs (miRs) profiles in nasopharyngeal carcinoma. METHOD: Screening the different expressions of miRs between nasopharyngeal carcinoma and the inflammatory tissues by the application of expression profiling of chip high-throughput and large-scale microarray analysis. Then we used RT-QPCR technology to prove the accuracy of screening results. RESULT: There were significant expression differences of miRs between nasopharyngeal carcinoma and the control tissues, 144 human miRs had 2 or more fold the difference ratio. Compared with the inflammatory tissues, we have found that miRs-34b, miRs-449b and miRs-7-1 significantly low expressed in nasopharyngeal carcinoma, yet miRs-125b, miRs-184, miRs-196b, miRs-205 and miRs-24-1 expressed high. The results were consistent with the microarray analysis. CONCLUSION: The difference expressed miRs might be closely related to the process of nasopharyngeal carcinoma, and the research on miRs profiles maybe provide a powerful target basis for early diagnosis and therapy of nasopharyngeal carcinoma.